Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Alazami syndrome: Report of three Indian patients with phenotypic spectrum from adolescence to adulthood.

Alazami syndrome (ALAZS) (MIM 615071) is a rare autosomal recessive disorder characterized by short stature, dysmorphic facial features, developmental delay, and impaired intellect. It was first reported in a Saudi Arabian family in 2012. Three Indian patients affected with ALAZS, one boy aged 13 years and other two sisters in their 40s are presented. These patients had few unreported dysmorphic facial features: high arched eyebrows and dental overcrowding. No microcephaly was noted in the sisters. One of the sisters did not have short stature. The boy also presented with unilateral buphthalmos of left eye. All three of them have been identified to harbor novel variants in LARP7.

Compound Heterozygosity for Hb Alperton (HBB: c.407C>T) and IVS-I-5 (G>C) (HBB: c.92+5G>C) Mutations Presenting as a Moderate Anemia in an Indian Family.

While knowledge of HBB gene mutations is necessary for offering prenatal diagnosis (PND) of ß-thalassemia (ß-thal), a genotype-phenotype correlation may not always be available for rare variants. We present for the first time, genotype-phenotype correlation for a compound heterozygous status with IVS-I-5 (G>C) (HBB: c.92+5G>C) and HBB: c.407C>T (Hb Alperton) mutations on the HBB gene in an Indian family. Hb Alperton is a very rare hemoglobin (Hb) variant with scant published information about its clinical presentation, especially when accompanied with another HBB gene mutation. Here we provide biochemical as well as clinical details of this variant.

WDR73 Mutations Cause Infantile Neurodegeneration and Variable Glomerular Kidney Disease.

Infantile-onset cerebellar atrophy (CA) is a clinically and genetically heterogeneous trait. Galloway-Mowat syndrome (GMS) is a rare autosomal recessive disease, characterized by microcephaly with brain anomalies including CA in some cases, intellectual disability, and early-infantile-onset nephrotic syndrome. Very recently, WDR73 deficiency was identified as the cause of GMS in five individuals. To evaluate the role of WDR73 mutations as a cause of GMS and other forms of syndromic CA, we performed Sanger or exome sequencing in 51 unrelated patients with CA and variable brain anomalies and in 40 unrelated patients with a diagnosis of GMS. We identified 10 patients from three CA and from two GMS families with WDR73 mutations including the original family described with CA, mental retardation, optic atrophy, and skin abnormalities (CAMOS). There were five novel mutations, of which two were truncating and three were missense mutations affecting highly conserved residues. Individuals carrying homozygous WDR73 mutations mainly presented with a pattern of neurological and neuroimaging findings as well as intellectual disability, while kidney involvement was variable. We document postnatal onset of CA, a retinopathy, basal ganglia degeneration, and short stature as novel features of WDR73-related disease, and define WDR73-related disease as a new entity of infantile neurodegeneration.

Experiences from Garbha-Swasthya helpline.

Garbha-Swasthya helpline is a telephone helpline run by a tertiary care private hospital to address issues related to pregnancy and its complications. A review of the helpline data from September 2010 to December 2012 was conducted to study caller characteristics, number of calls and related information, and the reasons to call. Out of the total 696 voice calls, 421 were new callers and 275 were repeat callers and they comprised mainly pregnant women (73.3%), their relatives (24.8%), and medical professionals (1.9%). Most calls were related to first pregnancy and were mainly from Pune and other cities of Maharashtra. All calls by the medical professionals were regarding drug safety in pregnancy. The commonest category of questions (27.4%) was about exposures (drugs, alcohol, eclipse, radiation, etc.) and preexisting medical illness followed by pregnancy complications (18.5%), common medical issues such as nausea/vomiting during pregnancy, and delivery and postpartum period (14% each). Although the total number of calls received has steadily increased, efforts to create public and professional awareness to increase the usage of this helpline are warranted.

Mutations in the human UBR1 gene and the associated phenotypic spectrum.

Johanson-Blizzard syndrome (JBS) is a rare, autosomal recessive disorder characterized by exocrine pancreatic insufficiency, typical facial features, dental anomalies, hypothyroidism, sensorineural hearing loss, scalp defects, urogenital and anorectal anomalies, short stature, and cognitive impairment of variable degree. This syndrome is caused by a defect of the E3 ubiquitin ligase UBR1, which is part of the proteolytic N-end rule pathway. Herein, we review previously reported (n = 29) and a total of 31 novel UBR1 mutations in relation to the associated phenotype in patients from 50 unrelated families. Mutation types include nonsense, frameshift, splice site, missense, and small in-frame deletions consistent with the hypothesis that loss of UBR1 protein function is the molecular basis of JBS. There is an association of missense mutations and small in-frame deletions with milder physical abnormalities and a normal intellectual capacity, thus suggesting that at least some of these may represent hypomorphic UBR1 alleles. The review of clinical data of a large number of molecularly confirmed JBS cases allows us to define minimal clinical criteria for the diagnosis of JBS. For all previously reported and novel UBR1 mutations together with their clinical data, a mutation database has been established at LOVD.

Lysosomal storage disorders identified in adult population from India: Experience of a tertiary genetic centre and review of literature.

Lysosomal storage disorders (LSDs) in adults have milder phenotype and variable age at presentation. Several studies have described the phenotype, genotype and treatment outcomes for adult-onset LSDs like Gaucher, Fabry, Pompe disease and others. We describe the first systematic study on the occurrence of LSDs in an adult population from India. It describes, the key clinical signs seen in these patients and those from literature review that can aid in early detection. Of 2102 biochemically diagnosed LSDs cases, 32 adult patients were identified with LSDs. Based on the clinical suspicion, screening test and enzyme study was carried out. Twenty-two patients were subjected to a genetic study to identify the causative variant in a respective gene. Of the 32 adult patients, we observed a maximum percentage of 37.5% (n = 12) cases with Gaucher disease, followed by 13% (n = 4) with Fabry disease. We found 10% of cases with MPS IVA and MPS I, and 9% cases with Pompe. Single case of adult mucolipidosis III and two cases each of Type 1 Sialidosis, Niemann-Pick disease B and metachromatic leukodystrophy were identified. We observed two common variants p.Leu483Pro and p.Ala487Thr in the GBA1 gene in 23% of Indian patients with adult Gaucher disease. No common variants were observed in other aforementioned LSDs. Study identified 50% of Fabry patients and 4% of Gaucher patients diagnosed at our centre to be adults. The prevalence of adult Pompe patients was low (3.4%) as compared to 80% reported in the Caucasian population. Adult LSDs such as, MPS III, GM1/GM2 gangliosidosis and Krabbe disease were not identified in our cohort.

Burden of rare genetic disorders in India: twenty-two years' experience of a tertiary centre.

BACKGROUND: Rare disorders comprise of ~ 7500 different conditions affecting multiple systems. Diagnosis of rare diseases is complex due to dearth of specialized medical professionals, testing labs and limited therapeutic options. There is scarcity of data on the prevalence of rare diseases in different populations. India being home to a large population comprising of 4600 population groups, of which several thousand are endogamous, is likely to have a high burden of rare diseases. The present study provides a retrospective overview of a cohort of patients with rare genetic diseases identified at a tertiary genetic test centre in India. RESULTS: Overall, 3294 patients with 305 rare diseases were identified in the present study cohort. These were categorized into 14 disease groups based on the major organ/ organ system affected. Highest number of rare diseases (D = 149/305, 48.9%) were identified in the neuromuscular and neurodevelopmental (NMND) group followed by inborn errors of metabolism (IEM) (D = 47/305; 15.4%). Majority patients in the present cohort (N = 1992, 61%) were diagnosed under IEM group, of which Gaucher disease constituted maximum cases (N = 224, 11.2%). Under the NMND group, Duchenne muscular dystrophy (N = 291/885, 32.9%), trinucleotide repeat expansion disorders (N = 242/885; 27.3%) and spinal muscular atrophy (N = 141/885, 15.9%) were the most common. Majority cases of ß-thalassemia (N = 120/149, 80.5%) and cystic fibrosis (N = 74/75, 98.7%) under the haematological and pulmonary groups were observed, respectively. Founder variants were identified for Tay-Sachs disease and mucopolysaccharidosis IVA diseases. Recurrent variants for Gaucher disease (GBA:c.1448T > C), ß-thalassemia (HBB:c.92.+5G > C), non-syndromic hearing loss (GJB2:c.71G > A), albinism (TYR:c.832 C > T), congenital adrenal hyperplasia (CYP21A2:c.29-13 C > G) and progressive pseudo rheumatoid dysplasia (CCN6:c.298T > A) were observed in the present study. CONCLUSION: The present retrospective study of rare disease patients diagnosed at a tertiary genetic test centre provides first insight into the distribution of rare genetic diseases across the country. This information will likely aid in drafting future health policies, including newborn screening programs, development of target specific panel for affordable diagnosis of rare diseases and eventually build a platform for devising novel treatment strategies for rare diseases.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis.

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs. -2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

Is germline transmission of MAD2 gene deletion associated with human fetal loss?

The spindle assembly checkpoint (SAC) monitors proper attachment of spindles to the kinetochore during mitotic and meiotic cell divisions and thus prevents aneuploidy. Chromosomal aneuploidy has been found to be associated with pregnancy loss and birth defects. Mad2 is one of the critical molecules of SAC. Deregulated Mad2 expression has been found to be associated with defective SAC-mediated abnormal meiotic progression in cell studies using animal models. Whether mutation in MAD2L1 is associated with the loss of Mad2 expression in aborted human fetuses is unknown. In this study, a correlation between aneuploidy and MAD2 defect was examined in primary fibroblast cultures obtained from abortuses. We report three trisomic abortuses with undetectable Mad2 expression. Further, quantitative real-time PCR revealed copy number deletion of MAD2 gene in these fetuses. Analysis of parental DNA samples available from two families revealed copy number loss of the same gene, suggesting Mendelian inheritance of MAD2 deletion. This germline transmission of exonic deletion of MAD2 is possibly associated with its loss of expression resulting in abnormal SAC function, subsequent aneuploidy and pregnancy loss.

Maternal one-carbon metabolism, MTHFR and TCN2 genotypes and neural tube defects in India.

BACKGROUND: Neural tube defects (NTDs) are among the most common severe congenital malformations, representing a long-term public health burden in India. A deranged one-carbon metabolism and genes regulating this metabolism have been linked to NTDs. Vitamin B(12) deficiency is reported to be more prevalent than folate deficiency in the Indian population. We investigated the role of maternal nutritional and genetic markers related to one-carbon metabolism in the etiology of NTDs. METHODS: We conducted a multicenter case-control study to compare plasma folate, vitamin B(12) , homocysteine and holo-transcobalamin levels, and polymorphisms in methylenetetrahydrofolate reductase (MTHFR, 677C>T, 1298A>C, 1781G>A and 236+724A>G) and transcobalamin (TCN2, 776C>G) genes, in 318 women with NTD-affected offspring (cases) and 702 women with apparently healthy offspring (controls). The samples were collected at diagnosis in cases and at delivery in controls. RESULTS: We observed a significant association of high maternal plasma homocysteine concentrations with NTDs in the offspring (p = 0.026). There was no association of maternal folate or B(12) levels with NTDs (p > 0.05) but low maternal holo-transcobalamin predicted strong risk of NTDs in the offspring (p = 0.003). The commonly associated maternal polymorphism 677C>T in the MTHFR gene did not predict risk of NTDs in the offspring (p > 0.05) and 1298A>C and 1781G>A polymorphisms in MTHFR were protective (p = 0.024 and 0.0004 respectively). Maternal 776C>G polymorphism in TCN2 was strongly predictive of NTD in the offspring (p = 0.006). CONCLUSION: Our study has demonstrated a possible role for maternal B(12) deficiency in the etiology of NTDs in India over and above the well-established role of folate deficiency.

Nutrigenetic determinants of neural tube defects in India.

JUSTIFICATION: Neural tube defects (NTDs) are one of the commonest birth defects with a high incidence in India. However, few studies have systematically looked into the etio-pathogeneis of NTDs, which mainly includes nutritional deficiencies and genetic predisposition. Efforts are afoot for universal food fortification with folic acid in the hope of preventing NTDs, without factual evidence of folate deficiency in the target population. EVIDENCE ACQUISITION: We conducted a review of Indian literature on NTDs focusing on the role of folate and vitamin B12 nutrition and common genetic polymorphisms in 1-carbon metabolism. We performed a literature search of Medline and Indian Medlars (www. indmed.nic.in) for articles using following search terms: Neural tube defect and India, published up to November 2008, on human subjects. We did not include individual case reports and case series describing surgical and medical management, genetic syndromes where NTD was only one of the features or unusual associations of NTDs with other clinical findings. RESULTS: Absence of a nationally representative large study, lack of interventional studies and methodological differences were conspicuous during this review. Larger studies are, therefore, urgently needed to delineate genenutrient interactions in association with NTDs in India. We urge that caution should be exercised before widespread folic acid fortification of food, without addressing the issue of concurrent B12 deficiency.

Bartsocas-Papas syndrome.

We report three sibs born to a third degree consanguineous Indian family affected with Bartsocas Papas Syndrome. All the three pregnancies were complicated by severe oligohydramnios, which is not commonly seen with Bartsocas-Papas syndrome.

Lysosomal Storage Disorders in Nonimmune Hydrops Fetalis (NIHF): An Indian Experience.

Lysosomal storage disorders (LSD) are rare inherited neurovisceral inborn errors of metabolism which may present as nonimmune hydrops fetalis (NIHF) during pregnancy. Although causes of NIHF are highly diverse, LSDs are one of the underlying causes of NIHF. The aim of this study was to elucidate most frequent causes of LSDs presenting as NIHF in Indian population. Several fetal tissues were investigated for enzymatic diagnosis of LSDs using modified fluorometric assays in the current prospective study carried out at our national tertiary center from 2006 through 2016. Other general causes of NIHF were ruled out. Twenty-one percent (7/33) of cases were confirmed to have LSDs. Two patients were diagnosed with Hurler syndrome; two had Sly syndrome and one each of Niemann-Pick disease type A/B, Gaucher's disease, and mucolipidosis. Four of eleven cases (36%) with recurrent NIHF were found to have LSDs. In spite of extreme rarity of LSDs, they should be considered as a potential cause of NIHF, especially with recurrent NIHF. Specific investigations of LSD leading to definitive diagnosis may aid the clinician in providing accurate genetic counseling and prenatal diagnosis to the patients and help in subsequent pregnancies to the families. Furthermore, early intervention and management with enzyme replacement therapy may be planned for the lysosomal storage disorders where available.

Maternal Serum Aneuploidy Screen and Adverse Pregnancy Outcomes.

OBJECTIVE: To find out whether maternal serum screening for fetal chromosomal aneuploidy predicts adverse pregnancy outcomes. METHODS: A two-year retrospective case-control study was conducted at a tertiary hospital. Pregnant women with a high-risk serum screen but with chromosomally normal fetuses (n = 189) were compared to those with low-risk screen (controls, n = 157) for adverse pregnancy outcomes. RESULTS: Women with high-risk double marker or combined screen were found to have higher prevalence of LBW [OR 2.56; 95 % CI (1.01-6.53), p < 0.05] and PT [OR 2.93; 95 % CI (1.11-7.65), p < 0.05], while women with high-risk triple screen had higher prevalence of PIH [OR 3.72; 95 % CI (1.23-11.18); p < 0.05], Oligo [OR 4.50; 95 % CI (1.30-15.64); p < 0.05], delivery by C-section [OR 2.51; 95 % CI (1.41-4.47); p < 0.005] as compared to low-risk women. PAPP-A was found to be a significant predictor of birth weight (R (2) = 12.2 %, ß ± SE = 0.224 ± 0.069; p < 0.005) and gestational age (R (2) = 4.9 %, ß ± SE = 0.613 ± 0.296; p < 0.05). Beta hCG in first and hCG in second trimester predicted oligohydramnios (R (2) = 9.2 %, ß ± SE = -0.077 ± 0.025; p < 0.005). The areas under the ROC curves of PAPP-A for LBW and PT were 0.70(p < 0.01) and 0.684 (p < 0.05), respectively. CONCLUSION: A "high-risk" maternal serum screen with abnormal PAPP-A and/or beta hCG/HCG is associated with adverse pregnancy outcomes and may help identifying women requiring additional fetal surveillance.

Many faces of Hirschsprung's disease.

Hirschsprung's disease (HSCR) is the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births. The etiology of HSCR is complex and is presumed to be a sex-influenced multifactorial disorder, with contributions from several genes. All the genes involved in HSCR are also involved with the early development of the enteric nervous system. HSCR is known to be associated with a chromosomal abnormality in 12 % of cases, and with other congenital anomalies in additional 18 % of cases. It is recommended that patients, including newborns, with HSCR undergo a careful assessment by a clinician trained in dysmorphology. Echocardiography, ultrasonography for urogenital malformations and skeletal x-rays should be routinely performed in cases with HSCR to rule out associated anomalies. HSCR associated with dysmorphic features or any additional systemic anomaly should prompt chromosomal studies. Genetic counseling should be provided to families of HSCR patients as the recurrence risk varies from 4 % to up to 50 % depending on whether it is non-syndromic or part of a specific syndrome.

Role of fetal autopsy as a complementary tool to prenatal ultrasound.

AIM: To correlate and compare prenatal ultrasound with fetal autopsy examination to detect structural births defects and provide specific diagnoses. METHODS: 141 second trimester fetuses (<20 weeks and <500 g) where pregnancy was terminated for structural birth defects and/or severe intra-uterine growth restriction (IUGR) or intra-uterine death, referred to our tertiary care private, teaching hospital were examined by a team of experienced pathologist and clinical geneticist. Findings of pathology examination were compared to those provided by ultrasound examination. RESULTS: A total of 301 structural abnormalities were noted. Specific etiology was identified or syndromic diagnosis was possible in 57/141 (40.4%) cases. The maximum number of systemic anomalies (45/301, 14.95%) was noted in the central nervous system (CNS). CNS anomalies were most commonly associated with facial dysmorphism including cleft lip/palate etc. There was a complete agreement between ultrasound and autopsy findings in 41/141 (29.07%) cases, additional information that did not influence the final diagnosis and/or counseling was obtained by autopsy in 65/1416 (46.09%) cases, while additional information that influenced the final diagnosis and/or counseling was provided by autopsy in 35/141 (24.82%) cases. CONCLUSION: Fetal autopsy serves as a complementary tool to fetal ultrasound due to its ability to pick up minor anomalies and/or anomalies that were missed on ultrasound. It may be routinely performed as an attempt to reach a specific diagnosis and offer appropriate counseling to couples, following pregnancy termination for fetal anomalies.

Burden of lysosomal storage disorders in India: experience of 387 affected children from a single diagnostic facility.

Lysosomal storage disorders (LSDs) are considered to be a rare metabolic disease for the national health forum, clinicians, and scientists. This study aimed to know the prevalence of different LSDs, their geographical variation, and burden on the society. It included 1,110 children from January 2002 to December 2012, having coarse facial features, hepatomegaly or hepatosplenomegaly, skeletal dysplasia, neuroregression, leukodystrophy, developmental delay, cerebral-cerebellar atrophy, and abnormal ophthalmic findings. All subjects were screened for I-cell disease, glycolipid storage disorders (Niemann-Pick disease A/B, Gaucher), and mucopolysaccharide disorders followed by confirmatory lysosomal enzymes study from leucocytes and/or fibroblasts. Niemann-Pick disease-C (NPC) was confirmed by fibroblasts study using filipin stain. Various storage disorders were detected in 387 children (34.8 %) with highest prevalence of glycolipid storage disorders in 48 %, followed by mucopolysaccharide disorders in 22 % and defective sulfatide degradation in 14 % of the children. Less common defects were glycogen degradation defect and protein degradation defect in 5 % each, lysosomal trafficking protein defect in 4 %, and transport defect in 3 % of the patients. This study demonstrates higher incidence of Gaucher disease (16 %) followed by GM2 gangliosidosis that includes Tay-Sachs disease (10 %) and Sandhoff disease (7.8 %) and mucopolysaccharide disorders among all LSDs. Nearly 30 % of the affected children were born to consanguineous parents and this was higher (72 %) in children with Batten disease. Our study also demonstrates two common mutations c.1277_1278insTATC in 14.28 % (4/28) and c.964G>T (p.D322Y) in 10.7 % (3/28) for Tay-Sachs disease in addition to the earlier reported c.1385A>T (p.E462V) mutation in 21.42 % (6/28).

Johanson-blizzard syndrome.

We present clinical features and genetic diagnosis in an Indian infant diagnosed with Johanson- Blizzard syndrome. This is a rare, autosomal recessive genetic condition with multi-system involvement and a characteristic facies. Molecular genetic testing is important to confirm the clinical diagnosis and offer prenatal diagnosis in future pregnancies.

An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3'- to 5'-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3'- to 5'-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis.